Metabolomic profiling of rare cell populations isolated by flow cytometry from tissues

Little is known about the metabolic regulation of rare cell populations because most metabolites are hard to detect in small numbers of cells. We previously described a method for metabolomic profiling of flow cytometrically-isolated hematopoietic stem cells (HSCs) that detects approximately 60 metabolites in 10,000 cells (Agathocleous et al., 2017). Here we describe a new method involving hydrophilic liquid interaction chromatography (HILIC) and high-sensitivity orbitrap mass spectrometry that detected approximately 160 metabolites in 10,000 HSCs, including many more glycolytic and lipid intermediates. We improved chromatographic separation, increased mass resolution, minimized ion suppression, extracted with acetonitrile, and eliminated sample drying. Most metabolites did not significantly change during cell preparation and sorting. We used this method to profile HSCs and circulating melanoma cells. HSCs exhibited increased glycerophospholipid metabolites relative to unfractionated bone marrow cells and altered purine biosynthesis after methotrexate treatment in vivo. Circulating melanoma cells were depleted for purine intermediates relative to subcutaneous tumors, suggesting they decrease purine synthesis during metastasis. These methods facilitate the routine metabolomic analysis of rare cell populations from tissues. Impact statement We developed a method for metabolomic analysis of small numbers of flow cytometrically isolated cells from rare cell populations such as hematopoietic stem cells and circulating cancer cells.